Stromal cell-derived factor (SDF-1) exists in many isoforms that are differentially distributed in various tissues. The highest expressed isoforms appear to be α and β which differ only at the carboxy terminus, with the β isoform being longer by four amino acid residues (72 vs. 68 residues). Both isoforms are found in circulation, but are differentially processed at the carboxy terminus by an unknown peptidase in serum. In vitro studies have shown that the terminal lysine of recombinant α isoform (K68) is immediately removed upon incubation in serum, whereas the recombinant β isoform is resistant to similar degradation. The amino termini of both isoforms, however, are vulnerable to cleavage by dipeptidyl peptidase IV (DPP-IV) with cleavage efficiencies (kcat/Km) higher than that of GLP-1. Similar to GLP-1, the removal of the two amino terminal residues inactivates SDF-1 by disruption of its receptor binding with CXCR4 and subsequent activation.
Recently, there has been increasing interest in the investigation of suppression of SDF-1 inactivation with DPP-IV inhibition. To date, however, most efforts have been limited to in vitro studies or in vivo studies with infused SDF-1 at superphysiological concentrations. These studies, though insightful, failed to provide direct evidence on suppression of endogenous SDF-1 inactivation by in vivo DPP-IV inhibition with therapeutic agents. The hindrance to such efforts is not the lack of specific assays that differentiate SDF-1 isoforms from their DPP-IV cleaved forms; many previous in vitro kinetics studies successfully employed mass spectrometry (MS) based methods to generate sequence specific measurements. The sensitivity of these MS methods, however, was not suitable for measurement of SDF-1 at endogenous levels (often sub nanomolar) in complex biological matrices such as plasma and serum. Instead, the endogenous SDF-1 isoforms in circulation were often quantified with antibody based immunoassays, which lacked the specificity to differentiate intact SDF-1 isoforms from DPP-IV cleaved ones and thus were inadequate for in vivo DPP-IV inhibition studies.